Antibody responses induced by the BNT162b2 mRNA COVID-19 vaccine in healthcare workers in a single community hospital in Japan.

INTRODUCTION: The effectiveness of several vaccines against coronavirus disease (COVID-19) has been reported in the real-world setting. However, it is still unknown how long antibodies persist following vaccination and whether or not the persistence of antibodies has a protective effect against COVID-19. METHODS: Healthcare workers who had received two doses of the BNT162b2 mRNA COVID-19 vaccine were enrolled, and a single-center study was conducted at the National Hospital Organization Hakodate National Hospital. Serum samples from all participants were collected 13-21 weeks (median: 20 weeks) after the second dose of vaccination. The antibody titers were measured using an electrochemiluminescence immunoassay (Elecsys® Anti-SARS-CoV-2 S). Data on characteristics of the participants were gathered from patient records and interview sheets. RESULTS: A total of 401 participants, among whom 70.1% were women and the median age was 42 years, were evaluated in this study. None of the participants had a definite COVID-19 history, and all participants who received complete vaccination showed positive antibody titers. The antibody titer was observed to be higher in participants with younger age (p < 0.001) and those who were females (p = 0.028). Despite the higher risk of infection than that of the general public, no vaccinated staff developed breakthrough infections. CONCLUSIONS: This study demonstrates the significant contribution of the BNT162b2 vaccine in the acquisition of anti-SARS-CoV-2S antibodies; therefore, the general population should benefit from these two vaccine doses, which are expected to be protective for at least five months.

Mucosal gene therapy using a pseudotyped lentivirus vector encoding murine interleukin-10 (mIL-10) suppresses the development and relapse of experimental murine colitis.

BACKGROUND: Therapeutic gene transfer is currently being evaluated as a potential therapy for inflammatory bowel disease. This study investigates the safety and therapeutic benefit of a locally administered lentiviral vector encoding murine interleukin-10 in altering the onset and relapse of dextran sodium sulfate induced murine colitis. METHODS: Lentiviral vectors encoding the reporter genes firefly-luciferase and murine interleukin-10 were administered by intrarectal instillation, either once or twice following an ethanol enema to facilitate mucosal uptake, on Days 3 and 20 in Balb/c mice with acute and relapsing colitis induced with dextran sulfate sodium (DSS). DSS colitis was characterized using clinical disease activity, macroscopic, and microscopic scores. Bioluminescence optical imaging analysis was employed to examine mucosal lentiviral vector uptake and transgene expression. Levels of tumor necrosis factor-α and interleukin-6 in homogenates of rectal tissue were measured by ELISA. Biodistribution of the lentiviral vector to other organs was evaluated by real time quantitative PCR. RESULTS: Mucosal delivery of lentiviral vector resulted in significant transduction of colorectal mucosa, as shown by bioluminescence imaging analysis. Lentiviral vector-mediated local expression of interleukin-10 resulted in significantly increased levels of this cytokine, as well as reduced levels of tumor necrosis factor-α and interleukin-6, and significantly reduced the clinical disease activity, macroscopic, and microscopic scores of DSS colitis. Systemic biodistribution of locally instilled lentiviral vector to other organs was not detected. CONCLUSIONS: Topically-delivered lentiviral vectors encoding interleukin-10 safely penetrated local mucosal tissue and had therapeutic benefit in this DSS model of murine colitis.

Cryptic transcripts from a ubiquitous plasmid origin of replication confound tests for cis-regulatory function.

A vast amount of research on the regulation of gene expression has relied on plasmid reporter assays. In this study, we show that plasmids widely used for this purpose constitutively produce substantial amounts of RNA from a TATA-containing cryptic promoter within the origin of replication. Readthrough of these RNAs into the intended transcriptional unit potently stimulated reporter activity when the inserted test sequence contained a 3' splice site (ss). We show that two human sequences, originally reported to be internal ribosome entry sites and later to instead be promoters, mimic both types of element in dicistronic reporter assays by causing these cryptic readthrough transcripts to splice in patterns that allow efficient translation of the downstream cistron. Introduction of test sequences containing 3' ss into monocistronic luciferase reporter vectors widely used in the study of transcriptional regulation also created the false appearance of promoter function via the same mechanism. Across a large number of variants of these plasmids, we found a very highly significant correlation between reporter activity and levels of such spliced readthrough transcripts. Computational estimation of the frequency of cryptic 3' ss in genomic sequences suggests that misattribution of cis-regulatory function may be a common occurrence.

Mixed invasive mucinous and non-mucinous adenocarcinoma of the lung with hematogenous metastases to multiple organs.

Mixed invasive mucinous and non-mucinous adenocarcinoma is a rare variant of lung adenocarcinoma. In pure invasive mucinous adenocarcinoma, multilobar and bilateral involvement are common, and extrathoracic metastasis is rare. Here, we report a case of mixed invasive mucinous and non-mucinous adenocarcinoma with distant metastasis to multiple organs without marked enlargement of the primary lung lesion. The pathological findings indicated high tumor invasiveness and the patient died 10 months after diagnosis despite chemoimmunotherapy. Further investigations are necessary to elucidate the clinical characteristics and appropriate management of mixed invasive mucinous and non-mucinous adenocarcinoma.

Design and selection of Toca 511 for clinical use: modified retroviral replicating vector with improved stability and gene expression.

Retroviral replicating vectors (RRVs) are a nonlytic alternative to oncolytic replicating viruses as anticancer agents, being selective both for dividing cells and for cells that have defects in innate immunity and interferon responsiveness. Tumor cells fit both these descriptions. Previous publications have described a prototype based on an amphotropic murine leukemia virus (MLV), encoding yeast cytosine deaminase (CD) that converts the prodrug 5-fluorocytosine (5-FC) to the potent anticancer drug, 5-fluorouracil (5-FU) in an infected tumor. We report here the selection of one lead clinical candidate based on a general design goal to optimize the genetic stability of the virus and the CD activity produced by the delivered transgene. Vectors were tested for titer, genetic stability, CD protein and enzyme activity, ability to confer susceptibility to 5-FC, and preliminary in vivo antitumor activity and stability. One vector, Toca 511, (aka T5.0002) encoding an optimized CD, shows a threefold increased specific activity in infected cells over infection with the prototype RRV and shows markedly higher genetic stability. Animal testing demonstrated that Toca 511 replicates stably in human tumor xenografts and, after 5-FC administration, causes complete regression of such xenografts. Toca 511 (vocimagene amiretrorepvec) has been taken forward to preclinical and clinical trials.

Invasive thymoma metastases to the pancreas: A case report.

INTRODUCTION AND IMPORTANCE: Thymoma is the most common type of tumor that develops in the thymic epithelial cells. Although thymomas can invade surrounding organs in the chest cavity, extrathoracic metastasis is very rare, and little is known about the prognosis and effective treatments for such tumors. Herein, we report a case of an invasive thymoma metastasizing to the pancreas after incomplete resection. CASE PRESENTATION: A 47-year-old man presented to our hospital with an anterior mediastinal mass. Although a thymic tumor was suspected, complete resection was not achieved because the tumor had invaded the pulmonary artery trunk, superior pulmonary vein, and left brachiocephalic vein. The pathological diagnosis was a Type B3 thymoma. The patient underwent chemotherapy and radiotherapy after surgery. Three years after surgery, computed tomography indicated a pancreatic mass suggestive of pancreatic cancer. Distal pancreatectomy was performed after neoadjuvant chemotherapy and radiotherapy. The pancreatic mass was diagnosed as Type B3 thymoma metastasis. Thirteen months after surgery for the pancreatic lesion, the patient underwent resection of the bilateral lung metastases. CLINICAL DISCUSSION: To the best of our knowledge, only four cases of metastatic thymic tumors in the pancreas have been reported. All patients who underwent surgical resection for pancreatic metastasis survived for >6 months including our case. CONCLUSION: In cases of thymic tumors with metastasis to extra-thoracic organs, complete resection of the metastatic lesions can contribute to prolonged survival.

Myasthenia Gravis Associated With Pembrolizumab for Relapsed Lung Cancer After Thymoma Resection.

Immunotherapy has demonstrated clinical efficacy in patients with thymic epithelial tumors; however, there is the potential risk of serious immune-related adverse events (irAEs). Here, we report a case of myasthenia gravis (MG) associated with pembrolizumab treatment that developed after thymoma resection in a patient with lung adenocarcinoma. Symptoms of MG occurred 16 days after pembrolizumab administration and progressed rapidly, necessitating mechanical ventilation and tracheostomy. Even after tumor resection, careful monitoring is crucial for patients with thymic tumors being managed with immune checkpoint therapy, particularly regarding the development of severe irAEs.

Antibody feedback contributes to facilitating the development of Omicron-reactive memory B cells in SARS-CoV-2 mRNA vaccinees.

In contrast to a second dose of the SARS-CoV-2 mRNA vaccine, a third dose elicits potent neutralizing activity against the Omicron variant. To address the underlying mechanism for this differential antibody response, we examined spike receptor-binding domain (RBD)-specific memory B cells in vaccinated individuals. Frequency of Omicron-reactive memory B cells increased ∼9 mo after the second vaccine dose. These memory B cells show an altered distribution of epitopes from pre-second memory B cells, presumably due to an antibody feedback mechanism. This hypothesis was tested using mouse models, showing that an addition or a depletion of RBD-induced serum antibodies results in a concomitant increase or decrease, respectively, of Omicron-reactive germinal center (GC) and memory B cells. Our data suggest that pre-generated antibodies modulate the selection of GC and subsequent memory B cells after the second vaccine dose, accumulating more Omicron-reactive memory B cells over time, which contributes to the generation of Omicron-neutralizing antibodies elicited by the third vaccine dose.

Small-sized peripheral squamous cell lung carcinoma with chest wall invasion.

In lung cancer, chest wall infiltration caused by a tumor with a small diameter is extremely rare. The pathophysiologic features and prognosis of this phenomenon are poorly understood. Here, we report on a case in which a small peripheral lung cancer showed marked invasion into the chest wall. Although complete resection and postoperative adjuvant treatment were performed, lymph node recurrence developed and the patient died in one and a half years. Peripheral lung cancer can show exophytic development and infiltration of the chest wall, leading to poor prognosis, even if the tumor size is relatively small.

Retroviral Replicating Vector Toca 511 (Vocimagene Amiretrorepvec) for Prodrug Activator Gene Therapy of Lung Cancer.

Therapeutic efficacy of retroviral replicating vector (RRV)-mediated prodrug activator gene therapy has been demonstrated in a variety of tumor models, but clinical investigation of this approach has so far been restricted to glioma and gastrointestinal malignancies. In the present study, we evaluated replication kinetics, transduction efficiency, and therapeutic efficacy of RRV in experimental models of lung cancer. RRV delivering GFP as a reporter gene showed rapid viral replication in a panel of lung cancer cells in vitro, as well as robust intratumoral replication and high levels of tumor transduction in subcutaneous and orthotopic pleural dissemination models of lung cancer in vivo. Toca 511 (vocimagene amiretrorepvec), a clinical-stage RRV encoding optimized yeast cytosine deaminase (yCD) which converts the prodrug 5-fluorocytosine (5-FC) to the active drug 5-fluorouracil (5-FU), showed potent cytotoxicity in lung cancer cells upon exposure to 5-FC prodrug. In vivo, Toca 511 achieved significant tumor growth inhibition following 5-FC treatment in subcutaneous and orthotopic pleural dissemination models of lung cancer in both immunodeficient and immunocompetent hosts, resulting in significantly increased overall survival. This study demonstrates that RRV can serve as highly efficient vehicles for gene delivery to lung cancer, and indicates the translational potential of RRV-mediated prodrug activator gene therapy with Toca 511/5-FC as a novel therapeutic strategy for pulmonary malignancies.

Splicing mediates the activity of four putative cellular internal ribosome entry sites.

A growing number of cellular mRNAs are thought to possess internal ribosome entry sites (IRESs), sequences that permit translation of a transcript independent of its 5' end and cap structure. Although dicistronic assays are the canonical method of testing sequences for IRES activity, they may produce false-positive results if unanticipated monocistronic RNAs arise from the dicistronic construct used. Using a dicistronic reporter system and a green fluorescent protein-tagged retrovirus to evaluate six previously reported cellular IRESs, we found that four contain 3' splice sites whose activity was required for apparent IRES function and which resulted in formation of monocistronic transcripts by splicing. Bioinformatic analysis revealed that the 3' splice sites identified in three of these putative IRESs are used in their native mRNAs and that the fourth is likely an artifactual sequence created during cDNA cloning. Our findings demonstrate a need for reexamination of other reported cellular IRESs by using careful RNA structural analysis to rule out splicing as the source of perceived IRES activity.

Optimization of enzyme-substrate pairing for bioluminescence imaging of gene transfer using Renilla and Gaussia luciferases.

BACKGROUND: Bioluminescence imaging (BLI) permits the non-invasive quantification and localization of transduction and expression by gene transfer vectors. The tendency of tissue to attenuate light in the optical region, however, limits the sensitivity of BLI. Improvements in light output from bioluminescent reporter systems would allow the detection of lower levels of expression, smaller numbers of cells and expression from deeper and more attenuating tissues within an animal. METHODS: With the goal of identifying substrates that allow improved sensitivity with Renilla luciferase (RLuc) and Gaussia luciferase (GLuc) reporter genes, we evaluated native coelenterazine and three of its most promising derivatives in BLI of cultured cells transduced with retroviral vectors encoding these reporters. Of the eight enzyme-substrate pairs tested, the two that performed best were further evaluated in mice to compare their effectiveness for imaging vector-modified cells in live animals. RESULTS: In cell culture, we observed striking differences in luminescence levels from the various enzyme-substrate combinations and found that the two luciferases exhibited markedly distinct abilities to generate light with the substrates. The most effective pairs were RLuc with the synthetic coelenterazine derivative ViviRen, and GLuc with native coelenterazine. In animals, these two pairs allowed similar detection sensitivities, which were eight- to 15-fold higher than that of the prototypical RLuc-native coelenterazine combination. CONCLUSIONS: Substrate selection can dramatically influence the detection sensitivity of RLuc and GLuc and appropriate choice of substrate can greatly improve the performance of reporter genes encoding these enzymes for monitoring gene transfer by BLI.

Primary undifferentiated pleomorphic sarcoma (Malignant fibrous histiocytoma) of the lung: A case report.

Primary UPS of the lung, so-called MFH, is a rare aggressive neoplasm and known to be a high risk of recurrence and metastasis. Surgical resection without residual tumor is the main option of treatment and the best chance for long-term survival.

Therapeutic efficacy of replication-competent retrovirus vector-mediated suicide gene therapy in a multifocal colorectal cancer metastasis model.

Replication-competent retrovirus (RCR) vectors are intrinsically incapable of infecting quiescent cells and have been shown to achieve highly efficient and tumor-restricted replicative spread and gene transfer in vivo after direct intratumoral injection in a variety of primary cancer models. However, i.v. delivery of RCR vectors expressing therapeutic genes has never previously been tested, particularly in an immunocompetent tumor model. Therefore, in the present study, we sought to test the therapeutic effect of an RCR vector (ACE-CD) carrying the yeast cytosine deaminase (CD) gene, which converts the nontoxic prodrug 5-fluorocytosine (5FC) into the chemotoxin 5-fluorouracil, after delivery by infusion into the locoregional circulation in a multifocal hepatic metastasis model of colon cancer. After confirmation of suicide gene cytotoxicity in vitro, multifocal hepatic tumors were established in syngeneic mice with murine CT26 colorectal cancer cells expressing firefly luciferase (CT26-Luc), and the ACE-CD vector was infused via intrasplenic injection into the portal circulation. Fourteen days after locoregional infusion, systemic administration of 5FC resulted in significant inhibition of bioluminescent signals in mice whose tumors had been infected with RCR but not in control mice. Notably, there was no detectable RCR vector spread to normal liver or bone marrow by quantitative PCR analysis. Our results thus show that locoregional delivery of a suicide gene by RCR vectors infused into the portal circulation results in progressive transduction of multiple tumor foci in the liver, without evidence of spread to adjacent normal parenchyma or extrahepatic tissues, and can achieve significant tumor growth inhibition.

Therapeutic activity of retroviral replicating vector-mediated prodrug activator gene therapy for pancreatic cancer.

Toca 511, a retroviral replicating vector (RRV) encoding the yeast cytosine deaminase (yCD) prodrug activator gene, which mediates conversion of the prodrug 5-fluorocytosine (5-FC) to the anticancer drug 5-fluorouracil (5-FU), is currently being evaluated in Phase II/III clinical trials for glioma, and showing highly promising evidence of therapeutic activity. Here we evaluated RRV-mediated prodrug activator gene therapy as a new therapeutic approach for pancreatic ductal adenocarcinoma (PDAC). RRV spread rapidly and conferred significant cytotoxicity with prodrug in a panel of PDAC cells. Efficient intratumoral replication and complete inhibition of tumor growth upon 5-FC administration were observed in both immunodeficient and immunocompetent subcutaneous PDAC models. Biodistribution of RRV was highly restricted in normal tissues, especially in immunocompetent hosts. Tumor growth inhibition by Toca 511 followed by 5-FC was also confirmed in the orthotopic PDAC model. This study provides the first proof-of-concept for application of Toca 511 and Toca FC (extended release 5-FC) to the treatment of human PDAC, and provided support for inclusion of PDAC in a Phase I study evaluating Toca 511 in various systemic malignancies, (NCT02576665), which has recently been initiated.

Tumor-selective gene expression in a hepatic metastasis model after locoregional delivery of a replication-competent retrovirus vector.

PURPOSE: Replication-competent retrovirus (RCR) vectors have been shown to achieve highly efficient and tumor-restricted replicative spread and gene transfer in vivo after direct intratumoral injection in a variety of primary cancer models. In this setting, the intrinsic inability of retroviruses to infect postmitotic normal cells, combined with their unique ability to persist through stable integration, allow further transduction of ectopic tumor foci as the infected cancer cells migrate. However, i.v. delivery of RCR vectors has never been tested previously, particularly in an immunocompetent tumor model. EXPERIMENTAL DESIGN: We combined optical imaging, flow cytometry, and molecular analysis to monitor RCR vector spread after administration via locoregional infusion in a hepatic metastasis model of colorectal cancer. RESULTS: Robust RCR replication was first confirmed in both human WiDr and murine CT26 colorectal cancer cells in vitro, with transduction levels reaching >90% in <12 days after virus inoculation at multiplicities of infection of 0.01 to 0.1. In vivo, infusion of RCR supernatant into the portal circulation resulted in progressive and significant transduction of multifocal intrahepatic CT26 tumors in syngeneic mice, averaging about 30% but with up to 60% transduction in some tumors within 4 weeks. However, immunohistochemistry and quantitative PCR analysis showed no evidence of RCR spread to adjacent normal liver or to any other normal tissues. CONCLUSIONS: Our results thus show that locoregional infusion of RCR vectors can be used to deliver therapeutic genes selectively to tumor cells in the liver while sparing normal hepatocytes and without dissemination to extrahepatic normal tissues.

Toca 511 gene transfer and treatment with the prodrug, 5-fluorocytosine, promotes durable antitumor immunity in a mouse glioma model.

BACKGROUND: Toca 511 (vocimagene amiretrorepvec) is a retroviral replicating vector encoding an optimized yeast cytosine deaminase (CD). Tumor-selective expression of CD converts the prodrug, 5-fluorocytosine (5-FC), into the active chemotherapeutic, 5-fluorouracil (5-FU). This therapeutic approach is being tested in a randomized phase II/III trial in recurrent glioblastoma and anaplastic astrocytoma (NCT0241416). The aim of this study was to identify the immune cell subsets contributing to antitumor immune responses following treatment with 5-FC in Toca 511-expressing gliomas in a syngeneic mouse model. METHODS: Flow cytometry was utilized to monitor and characterize the immune cell infiltrate in subcutaneous Tu-2449 gliomas in B6C3F1 mice treated with Toca 511 and 5-FC. RESULTS: Tumor-bearing animals treated with Toca 511 and 5-FC display alterations in immune cell populations within the tumor that result in antitumor immune protection. Attenuated immune subsets were exclusive to immunosuppressive cells of myeloid origin. Depletion of immunosuppressive cells temporally preceded a second event which included expansion of T cells which were polarized away from Th2 and Th17 in the CD4+ T cell compartment with concomitant expansion of interferon gamma-expressing CD8+ T cells. Immune alterations correlated with clearance of Tu-2449 subcutaneous tumors and T cell-dependent protection from future tumor challenge. CONCLUSIONS: Treatment with Toca 511 and 5-FC has a concentrated effect at the site of the tumor which causes direct tumor cell death and alterations in immune cell infiltrate, resulting in a tumor microenvironment that is more permissive to establishment of a T cell mediated antitumor immune response.

Retroviral replicating vector-mediated gene therapy achieves long-term control of tumor recurrence and leads to durable anticancer immunity.

BACKGROUND: Prodrug-activator gene therapy with Toca 511, a tumor-selective retroviral replicating vector (RRV) encoding yeast cytosine deaminase, is being evaluated in recurrent high-grade glioma patients. Nonlytic retroviral infection leads to permanent integration of RRV into the cancer cell genome, converting infected cancer cell and progeny into stable vector producer cells, enabling ongoing transduction and viral persistence within tumors. Cytosine deaminase in infected tumor cells converts the antifungal prodrug 5-fluorocytosine into the anticancer drug 5-fluorouracil, mediating local tumor destruction without significant systemic adverse effects. METHODS: Here we investigated mechanisms underlying the therapeutic efficacy of this approach in orthotopic brain tumor models, employing both human glioma xenografts in immunodeficient hosts and syngeneic murine gliomas in immunocompetent hosts. RESULTS: In both models, a single injection of replicating vector followed by prodrug administration achieved long-term survival benefit. In the immunodeficient model, tumors recurred repeatedly, but bioluminescence imaging of tumors enabled tailored scheduling of multicycle prodrug administration, continued control of disease burden, and long-term survival. In the immunocompetent model, complete loss of tumor signal was observed after only 1-2 cycles of prodrug, followed by long-term survival without recurrence for >300 days despite discontinuation of prodrug. Long-term survivors rejected challenge with uninfected glioma cells, indicating immunological responses against native tumor antigens, and immune cell depletion showed a critical role for CD4+ T cells. CONCLUSION: These results support dual mechanisms of action contributing to the efficacy of RRV-mediated prodrug-activator gene therapy: long-term tumor control by prodrug conversion-mediated cytoreduction, and induction of antitumor immunity.

CD4+ and CD8+ T cells cooperate to improve prognosis of patients with esophageal squamous cell carcinoma.

The purpose of this study is to clarify the roles of immune cell types, both individually and synergistically, in esophageal squamous cell carcinoma (ESCC). One hundred and twenty-two patients (105 males and 17 females; mean age, 62.3 years) with primary ESCC underwent surgical tumor resection at the Department of Surgical Oncology, School of Medicine, Hokkaido University and two affiliated hospitals between 1989 and 1999. Immunohistochemical analyses were performed for CD4, CD8, and CD57 (surface markers for natural killer cells). Patient prognosis was found to correlate with the number of CD4(+) and CD8(+) T cells in the stroma and the number of CD8(+) T cells within the cancer cell nest. Furthermore, the number of CD8(+) T cells in the stroma and within the cancer cell nest was found to be correlated [correlation coefficient (r) = 0.790; P < 0.0001). However, no correlation was observed between the number of natural killer cells and patient prognosis. Patients were classified into the following four groups based on CD4(+) and CD8(+) T-cell count: CD4/8(+/+), CD4/8(+/-), CD4/8(-/+), CD4/8(-/-). For the general patient pool, as well as for selected p-stage III and IV cases (n = 48), the survival rate for CD4/8(+/+) patients was significantly higher than that for the other three groups (log-rank test, P = 0.0012 and 0.0088, respectively). Multivariate analysis identified CD4/8(+/+) status, T classification, and N classification as independent prognostic factors. In conclusion, cooperation between CD4(+) and CD8(+) T cells correlates strongly with ESCC patient prognosis.

CD40 Expression in Human Esophageal Squamous Cell Carcinoma Is Associated with Tumor Progression and Lymph Node Metastasis.

BACKGROUND: The co-stimulatory molecule cluster of differentiation 40 (CD40) is widely expressed in various types of malignant tumors, but its role remains unclear. The purpose of this study was to investigate the relationship between CD40 expression and clinicopathological variables in patients with esophageal squamous cell carcinoma (ESCC), as well as the function of CD40 expressed on ESCC tumor cells in vitro. MATERIALS AND METHODS: Tumor specimens of patients who underwent surgical resection for ESCC were immunohistochemically analyzed for CD40 expression. RESULTS: Of the 122 specimens, 45 (37%) were positive for CD40. Significant positive correlation was found between CD40 expression and p-stage (p=0.0011), histopathological grade (p=0.0143), pT-classification (p=0.0011), and pN-classification (p=0.0007). Survival of patients with stage III and IV disease with positive CD40 expression was significantly shorter than that of those with negative expression (log-rank test, p=0.0422). In in vitro analysis, while the addition of recombinant human CD154 did not inhibit growth, it did induce a significant increase in interleukin 6 production in ESCC cell lines. CONCLUSION: These results suggest that functional expression of CD40 on tumor cells might play an important role in tumor progression and lymph node metastasis in ESCC.